Method for detecting mutations using an URA3 reporter gene

Abstract

A method for detecting nonsense mutations and frameshift mutations in a test nucleic acid fragment which is simple and may be used even when the size of the test nucleic acid fragment is large is disclosed. The method involves inserting the test nucleic acid fragment into a vector containing a promoter, a translational initiation codon, and a reporter gene, and transforming a host cell with the vector to express a fusion polypeptide. The production of the fusion polypeptide is assayed for in the host cell, and non-production of the fusion polypeptide is indicative of a nonsense and/or frameshift mutation in the test nucleic acid fragment.

What is claimed is:  
     
       1. A method for detecting nonsense mutations and frameshift mutations in a test nucleic acid fragment, comprising the steps of: 
       inserting a test nucleic acid fragment which may contain a frameshift mutation or a nonsense mutation or both into a site of a vector having a promoter, a translational initiation codon downstream of said promoter, a reporter gene which is a structural gene located downstream of said translational initiation codon, which is operably linked to said promoter, which encodes a polypeptide, in cases where a fusion polypeptide is formed, said fusion polypeptide being detectable based on a function of said polypeptide encoded by said reporter gene, said site into which said test nucleic acid fragment is inserted being located downstream of said translational initiation codon and upstream of said reporter gene, said test nucleic acid fragment being one which allows, when inserted, in-frame location of said reporter gene with respect to said translational initiation codon when said test nucleic acid is normal type;  
       expressing said test nucleic acid fragment and said reporter gene downstream thereof in the resulting recombinant vector in a host cell; and  
       determining whether said fusion polypeptide having said function of said polypeptide encoded by said reporter gene is produced or not, non-production of said fusion protein having said function of said polypeptide encoded by said reporter gene being indicative that said test nucleic acid contains a nonsense mutation or a frameshift mutation or both,  
       wherein said host cell is selected from the group consisting of: an auxotrophic host cell, a temperature-sensitive host cell, a pH-sensitive host cell, and a drug-sensitive host cell and said reporter gene is Uracil 3 (URA3) gene.  
     
     
       2. The method according to  claim 1 , wherein said translational initiation codon is contained in a second structural gene located upstream of said reporter gene and said test nucleic acid fragment is inserted between said second structural gene and said reporter gene and said second structural gene is a hemagglutinin gene. 
     
     
       3. The method according to  claim 1  or  2 , wherein said host cell is a yeast cell. 
     
     
       4. The method according to  claim 3 , wherein said yeast is  Saccharomyces cerevisiae.    
     
     
       5. The method according to  claim 1  or  2 , wherein said test nucleic acid fragment is Breast Cancer 1 (BRCA1) gene or a fragment thereof. 
     
     
       6. The method according to  claim 1  or  2 , wherein said test nucleic acid fragment is Adenomatous Polyposis Coli (APC) gene or a fragment thereof. 
     
     
       7. The method according to  claim 1  or  2 , wherein said test nucleic acid fragment is Breast Cancer 2 (BRCA2) gene or a fragment thereof. 
     
     
       8. The method according to  claim 1  or  2  wherein said test nucleic acid fragment is Human MutS Homologue 2 gene or a fragment thereof.